Project Summary Pseudomonas aeruginosa (Pa) is a major human pathogen whose virulence is predicated upon its ability to form biofilms - slimy layers of polysaccharides and bacteria that allow Pa to colonize wounds and to evade antibiotics. My lab has recently uncovered novel roles for bacteriophages in the formation and function of Pa biofilms. We reported that Pf bacteriophages (Pf phages) produced by Pa spontaneously assemble the loose network of host and microbial polymers present at sites of Pa infection into a dense, highly stable, liquid crystal. This crystalline architecture enhances biofilm adhesiveness, viscosity, and resistance to desiccation. We also recently reported that Pf phage and this crystalline architecture contribute to antibiotic tolerance by binding and sequestering antibiotics. This effect is dependent on the charge properties of the antibiotic; positively charged antibiotics, like tobramycin, are efficiently sequestered within crystalline bundles of negatively charged phage while neutrally charged antibiotics, like ciprofloxacin, are not. These data suggest that presence of Pf phage may be an important factor in determining which antibiotics are effective against Pa. Together, these data suggest that Pa bacteria and Pf phage may partner in ways that contribute to the pathogenicity of biofilm infections. Consistent with this, our preliminary data suggest that, in a murine model of Pa biofilm wound infection, the presence of Pf phage contributes to chronic infection. These novel insights into the pathogenic contributions of Pf phage have the potential to open up a new front in the fight against Pa biofilms. However, first it is important to determine their relevance to human chronic wound infections. In Aim 1, we will assess whether Pa biofilms in human wounds have crystalline structure and define the relationship between Pf phage levels and clinical outcomes, including antibiotic resistance. Give these contributions to biofilm pathogensis, it may be beneficial to to target Pf phage therapeutically. To this end, we have developed monoclonal antibodies and vaccines directed against the Pf phage coat protein, CoaB. Our initial experiments suggest that antibodies can interfere with biofilm organization and promote antibiotic penetrance in vitro. This suggests that it may be possible to prevent Pa wound infections by immunizing diabetic individuals against Pf phage. However, first it is necessary to demonstrate the impact of these treatments in vivo. In Aim 2, we will test whether antibodies directed against Pf phage promote antibiotic efficacy in a mouse model of Pa biofilm infection. Together, these aims represent a bold and radically novel approach to Pa biofilm infections and chronic wound infections. If successful, the data generated from these studies will support investigations into the role of Pf phage in the pathophysiology of wound infections and novel therapies targeting Pf phage.